Temporal dynamics of carbon flow through the microbial plankton

MARINE ECOLOGY PROGRESS SERIESMar Ecol Prog Ser

Vol. 461: 31–46, 2012doi: 10.3354/meps09782

Published August 8

INTRODUCTION

Many productive pelagic environments have multi -vorous food webs, in which both classic herbivorousfood chains (large phytoplankton, suspension-feed-

ing zooplankton and fish) and microbial trophic com-ponents (heterotrophic bacteria, cyanobacteria, smalleukaryotic algae and protozooplankton) contributesignificantly to carbon fluxes. Thus, the fate of pri-mary production with regard to carbon cycling

© Inter-Research 2012 · www.int-res.com*Email: [email protected]

Temporal dynamics of carbon flow through the microbial plankton community in a coastal

upwelling system off northern Baja California, Mexico

Lorena Linacre1, 2,*, Michael R. Landry3, Ramón Cajal-Medrano4, J. Rubén Lara-Lara5, J. Martín Hernández-Ayón6, Rosa R. Mouriño-Pérez2,

Ernesto García-Mendoza5, Carmen Bazán-Guzmán5

1Programa de Doctorado en Oceanografía Costera, Facultad de Ciencias Marinas/Instituto de Investigaciones Oceanológicas, 4Facultad de Ciencias Marinas, and 6Instituto de Investigaciones Oceanológicas;

Universidad Autónoma de Baja California (UABC), Ensenada, Baja California 22860, Mexico2Departamento de Microbiología, División de Biología Experimental y Aplicada, and

5Departamento de Oceanografía Biológica, División de Oceanología; Centro de Investigación Científica y de EducaciónSuperior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico

3Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093-0227, USA

ABSTRACT: We investigated the temporal dynamics of carbon flow through the microbial food webof a coastal upwelling system (ENSENADA station) off northern Baja California during 6 cruises(September 2007 to November 2008). Carbon biomass assessments for major autotrophic size groups(pico- to micro-sized cells) and their microzooplankton grazers were based on analyses using flowcytometry, HPLC pigments and epifluorescence microscopy. Taxon-specific phytoplankton growthand microzooplankton grazing rates were determined from 24 h in situ incubations in the euphoticzone using an abbreviated 3-treatment dilution technique. Carbon biomass and instantaneousgrowth and grazing rate determinations were used to estimate daily rates of taxon- specificproduction and losses due to microzooplankton grazing. Overall, microbial biomass showed a closebalance between autotrophic and heterotrophic components, except during a period of very strongupwelling (April 2008), which favored large phytoplankters and high primary production. Through-out a wide range of environmental conditions, the community primary production (PP) attributedboth to small (mostly picophytoplankton and prasinophytes) and large (mostly diatoms and au-totrophic dinoflagellates) autotrophs was significantly grazed (78 ± 9% of PP) by small (<20 µm) andlarge (>20 µm) ciliates and flagellates (including mixotrophic dinoflagellates), respectively, showingcomplementary temporal shifts in protistan grazer types that matched the dominant phytoplankton.While large diatoms were strongly consumed by large ciliates during the 2 most productive periods(September 2007 and April 2008), pico- and nano-sized phytoplankton were grazed most by nanofla-gellates and small ciliates from November 2007 to January 2008. Consequently, biogenic carbon pro-duction in this ecosystem is transferred through a multivorous food web.

KEY WORDS: Biogenic carbon flow · Multivorous food web · Phytoplankton growth rate · Phytoplankton grazing rate · Primary production

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Mar Ecol Prog Ser 461: 31–46, 2012

within the upper layer or export to the deep oceandepends on temporal variability in the relativestrengths of these alternate trophic pathways (Le -gendre & Rassoulzadegan 1995, 1996).

Within the microbial food web, nano- to micro-sized grazers <200 µm in size (here defined collec-tively as the microzooplankton) are ubiquitous andmajor consumers of small and large primary produc-ers as well as heterotrophic bacteria (Sherr & Sherr2002, Calbet 2008). To quantify the general magni-tude of the phytoplankton-microzooplankton trophiclink, experimental studies of phytoplankton growthand microzooplankton grazing have been conductedin a variety of oceanic habitats using the dilutiontechnique (Landry & Hassett 1982). On average,microzooplankton consume ~67% of phytoplanktondaily production over a broad range of habitat types(Calbet & Landry 2004). Most of the remaining netproduction is believed to be grazed by mesozoo-plankton. The mean estimate for mesozooplanktongrazing loss is 22.6% of primary production (PP), butit can vary substantially by system trophic state (Cal-bet 2001). Recent studies that have examined thegrazing contributions of both micro- and mesozoo-plankton in open-ocean and coastal systems haveshown that the difference between measured pro-duction and the combined losses to the 2 grazer sizefractions largely explain the net daily changes inphytoplankton biomass observed in the environment(Landry et al. 2009, 2011).

Because upwelling regions are highly productiveand potentially important in transferring carbon fromcontinental shelves to slopes (Walsh et al. 1981),more knowledge of the magnitude and temporalvariability of pelagic food web activity in theseregions is needed. Under the contrasting environ-mental conditions that drive the production dynamicsof such systems, carbon fluxes are strongly respon-sive to the dominant size structure and compositionof the phytoplankton community (Latasa et al. 2005,Gutiérrez-Rodríguez et al. 2010) and to the balancebetween the phytoplankton production and its graz-ing loss due to consumers of different types and sizes.Thus, while coastal upwelling areas are typicallyviewed as diatom dominated, implying strong link-ages to suspension-feeding metazoans and higherlevel consumers, substantial portions of the systemproductivity can be channeled through smaller auto-trophs and microbial pathways, including direct con-sumption of diatoms by protists (Neuer & Cowles1994, Landry et al. 2000a, 2009, Stelfox-Widdicombeet al. 2004, Aberle et al. 2007, Sherr & Sherr 2007,Vargas et al. 2007, Teixeira et al. 2011). Rapid re -

sponse of the protistan grazer assemblage to largesystem swings in the biomass and composition ofphytoplankton prey promotes a planktonic food webcharacterized by efficient utilization of primary pro-duction and carbon and nutrient cycling within theeuphotic zone (Calbet & Landry 2004).

In the coastal upwelling region off western BajaCalifornia (WBC), picoplankton populations (auto -trophic and heterotrophic) contribute significantly tocarbon fluxes, with temporal dynamics strongly influ-enced by seasonal oceanographic conditions (Linacreet al. 2010a,b). In the present study, we combinemicroscopic and flow cytometric (FCM) assessmentsof community biomass and composition with experi-mental studies of growth and grazing rates to gain abroader perspective of carbon-based production andgrazing for different phytoplankton groups. Our general hypothesis is that a strong coupling existsbetween phytoplankton carbon production and graz-ing losses to microzooplankton, which involves com-plementary shifts in protistan grazer types to matchthe temporal variability of dominant phytoplankton.In this context, our main goal is to evaluate the contribution of different phytoplankton groups tocommunity biomass, production and grazing underdiffering environmental conditions. Our results high-light the general multivorous nature of carbon fluxesthrough the microbial plankton community in thiscoastal upwelling system.

MATERIALS AND METHODS

Sampling and experimental design

Experimental studies of phytoplankton growth andprotistan grazing were conducted at ENSENADAstation (31° 40.105’ N, 116° 41.596’ W) in the northernregion off WBC (Fig. 1) as part of the FLUCAR (Car-bon Sources and Sinks in the Continental Margins ofthe Mexican Pacific Waters) project. During theperiod from 24 September 2007 to 11 November2008, 6 sets of experiments were incubated in situfor 24 h at the station. Following the experimentalapproach of Landry et al. (2008), we used an abbrevi-ated 3-treatment dilution protocol (100, 30 and 10%of whole seawater, without an addition of nutrientsand non-replicated) to estimate rates of phytoplank-ton growth and microzooplankton grazing. Based onin situ fluorescence profiles, seawater for the experi-ments was collected in 5 l Niskin bottles from theeuphotic zone at the subsurface chlorophyll maxi-mum (SCM) and from the depths above and below

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Linacre et al.: Carbon flow through a microbial plankton community

the SCM. Details of the experimental design aredescribed by Linacre et al. (2010a). Briefly, dilutiontreatments (10, 30 and 100% of natural planktondensity) were prepared in clear polycarbonate bot-tles (2 l), mixing a whole seawater fraction with waterdirectly filtered from the Niskin bottles using a peri-staltic pump, silicone tubing and an in-line Suporcapfilter capsule. The bottles were tightly capped,placed into net bags, secured with snap hooks to aweighted line hanging from surface floats andattached to a fixed buoy at the depths of initial sam-ple collection. The experiments were conducted inthe early morning and deployed prior to sunrise.

Picoplankton analyses

Samples were taken for FCM analyses at the startand end of each experiment to determine initialabundances and to compute carbon biomass and cell-specific growth and grazing loss rates in the dilutionincubations. For enumeration of picophytoplanktonand heterotrophic bacteria, 2 ml samples were pre-served (in 0.5% paraformaldehyde, final concentra-tion), flash frozen and stored in liquid nitrogen untilanalysis. Samples were analyzed with a Beckman-Coulter Altra cytometer following the approach de-scribed by Linacre et al. (2010a) and Taylor et al.(2011). The cell abundances of hetero trophic bacteria

(H-Bact), Prochlorococcus spp. (Pro), Synechococcusspp. (Syn) and pico eukaryotes (P-Euks) were con-verted to carbon biomass based on carbon per cellcon versions estimated for each category by cruiseand depth using bead- normalized forward-anglelight scattering (FALS) to assess cell biovolume (BV)variability. For each cell category, we assumed amean cell carbon estimate from literature values 20,39, 82 and 1000 fg C cell−1 for H-Bact, Pro, Syn and P-Euks, respectively (Lee & Fuhrman 1987, Worden etal. 2004, Sherr et al. 2005). We then used the FALS ra-tio (FALSsample: FALSmean)0.55 as a scaling factor for thecell size variability of each population (Linacre et al.2010a and literature therein). According to these cal-culations, the ranges in cell carbon estimates were 16to 24 (H-Bact), 34 to 47 (Pro), 64 to 119 (Syn) and 608to 1421 fg C cell−1 (P-Euks). For size structure assess-ments, we assumed that the largest ‘pico’ sized cellswere 2 µm in diameter, although some in the enumer-ated P-Euks category may have been larger than thisformal cut-off (Linacre et al. 2010a).

Pigment analyses

Seawater samples (1 l) were collected for pigmentanalysis at the start and end of each experiment.Samples were filtered through 25 mm diameter glassfiber filters (GF/F) and frozen immediately in liquidnitrogen until analysis. Pigment extraction was con-ducted following the procedures described byAlmazán- Becerril & García-Mendoza (2008). Filterswere placed in 2 ml capped tubes with 0.5 mm dia -meter zirconia/ glass beads in 1 ml of cold acetone(100%), vortexed and stored at 4°C in a processrepeated 3 times. The samples were cleaned by centrifugation. Pigments were quantified using highperformance liquid chromatography (HPLC) as inVan Heukelem & Thomas (2001), as modified byColom bo-Pallotta et al. (2006). The samples wereanalyzed in a Shimadzu AV-10 series HPLC instru-ment equipped with a Zorbax Eclipse XDBC-8reverse phase column (150 mm × 4.6 mm internaldiameter, 3.5 µm particles, 60°C). An absorptiondetector was set up at 436 nm. Pigment peaks in thechromatograms were identified by comparison of theretention times with those of pure standards andextracts prepared from algal cultures of known pig-ment composition. This protocol achieves baselineseparation of monovinyl chlorophyll a (MVChl a) anddivinyl chlorophyll a (DVChl a) and was calibratedwith 16 pigment standards (DHI; www.labproducts.dhigroup.com). Standard purity determination and

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Fig. 1. Location of ex pe ri -mental site ‘ENSENADAstation’ (31° 40.105’ N,116° 41.596’ W) in thenor thern coas tal watersof western Baja Cali-

fornia, Mexico


the calibration protocol were as in Wright & Man-toura (1997).

Taxonomic assignments for the major pigmentsmeasured were based on known pigment composi-tions (Jeffrey & Vesk 1997, Wright & Jeffrey 2006).Significant peaks of chlorophyllide a (Chlide a), wereseen in the chromatograms during the September2007 and April 2008 cruises, possibly due to filtrationdamage of the abundant large diatom chains duringthose seasons (Wright & Mantoura 1997). Therefore,total chlorophyll a (TChl a), a proxy for the totalautotrophic community, was composed of MVChl a +DVChl a + Chlide a (for the September 2007 andApril 2007 cases), following the approach of Latasa &Bidigare (1998). MVChl a is found in all eukaryoticphytoplankton and in the photosynthetic prokaryoteSynechococcus spp. DVChl a, only weakly detectedin our HPLC analyses, is a specific marker for Pro -chloro coccus. The pigments 19’-hexanoyloxyfuco-xanthin (Hex-Fuco) and 19’-butanoyloxyfucoxanthin(But-Fuco) are found in both prymnesiophytes andpelagophytes, but Hex-Fuco occurs in higher con-centrations in prymnesiophytes, while But-Fuco(more weakly detected in our samples) is the domi-nant accessory pigment of pelagophytes. Fucoxan-thin (Fuco) was assumed in the present study to bemostly indicative of diatoms, although it can alsobe found in prymnesiophytes and pelagophytes.Prasinoxanthin (Prasinox) is found in some types ofprasinophytes, and alloxanthin (Allox) is an unam-biguous marker for cryptophytes. Both pigmentswere de tec ted at moderate levels in our samples.Peridinin (Perid) is found only in dinoflagellates,although it is absent or very low in many taxa. Thisbiomarker was occasionally detected in the presentstudy, but it contributed minimally to the total phyto-plankton community pigments (Jeffrey & Vesk 1997,Wright & Jeffrey 2006).

Microscopic assessments of nano- and microplankton

Estimates of carbon biomass for size classes andfunctional groups of autotrophic and heterotrophiceukaryotes (grazers) were made by digitally en -hanced epifluorescence microscopy on 2 slide prepa-rations, after freezing and storage at −30°C, using themethodology described by Taylor et al. (2011). Cells<10 µm in size were enumerated in seawater samplesof 50 ml, preserved with paraformaldehyde (0.5%final concentration), stained with proflavin (0.33%w/v) and DAPI (10 µg ml−1) and mounted onto

0.8 mm black Nuclepore filters. Larger cells wereenumerated from 500 ml samples, preserved with260 µl of alkaline Lugol’s solution followed by 10 mlof buffered formalin and 500 µl of sodium thiosulfate(modified protocol from Sherr & Sherr 1993), stainedwith proflavin (0.33% w/v) and DAPI (10 µg ml−1)and mounted onto 8 µm black Nuclepore filters. Theslides were imaged and digitized at 630× (50 ml) and200× (500 ml) using a Zeiss AxioVert 200M invertedepifluorescence microscope with an AxioCam HRblack/ white digital camera. Cell BV (µm3) weredetermined from length (L) and width (W) measure-ments using the formula for a prolate sphere (BV =0.524 × L × W × H), where cell height (H) on the filterswas empirically determined to be 0.5 W for nakedflagellates (including dinoflagellates) (Taylor et al.2011). Carbon (C; pg cell−1) biomass was computedfrom BV from the equations of Menden-Deuer &Lessard (2000): C = 0.216 × BV0.939 for non-diatoms,and C = 0.288 × BV0.811 for diatoms.

For each experiment, 250 ml samples were alsofixed with 5% acid Lugol’s solution for a biomassestimation of ciliates (Cil), which were sub-optimallypreserved by the other methods and rarely countedon the slides. Subsamples of 10 ml (for September2007 and April 2008) and 50 ml (for November2007/08, January 2008 and August 2008) were set-tled in Utermöhl sedimentation chambers for at least24 h, counted, sorted by size class to >20 and <20 µmand measured at 400× with a Zeiss AxioVert 200inverted microscope equipped with an AxioCam HRccolor digital camera (Microbiology Department,CICESE). BV (µm3) estimates were based on meas-ured L and W dimensions and the closest geometricshapes for individual cells (50 to 100 cells) collectedfrom the SCM. To convert cell BV estimates to car-bon, we used the equations C (µg) = 0.12 + 0.19 BV(µm3) for naked ciliates (Putt & Stoecker 1989) and C(pg) = 44.5 + 0.053 lorica volume (µm3) for loricate ciliates (Verity & Langdon 1984). Median cell carbonestimates for each of the >20 and <20 µm ciliate sizeclasses were used to calculate ciliate carbon biomassfor each cruise and depth sample.

Microzooplankton grazers in this coastal systemwere mainly represented by Cil and heterotrophicand mixotrophic flagellates (dinoflagellates in cluded;H-Flag and H-Dino). To represent the potential graz-ing contribution of mixotrophic groups, we followedthe approach of Landry et al. (2011), based on theempirical results of Stukel et al. (2011), and countedhalf of the biomass of autotrophic flagellates (A-Flag,including dinoflagellates) as contributors to grazingon phytoplankton.

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Growth and grazing estimates

Instantaneous rates of phytoplankton growth (μ)and mortality loss (m) due to protistan grazers wereestimated from dilution incubations in accordancewith Landry & Hassett (1982), using the abbreviated3-treatment dilution protocol described by Linacre etal. (2010a). Initial pigment concentrations and popu -lation abundances (C0) from HPLC and FCM ana -lyses, respectively, were determined for each dilutiontreatment from measured concentrations in the un -filtered seawater (100%) and the proportion of unfil-tered (Di) seawater in the treatment i. Final concen-trations (Ct) were measured in each bottle at the endof the 24 h incubations (t). Daily net-specific rates ofchange were estimated as ki = ln(Ct/C0)/t, where C0

and Ct are expressed as µg C l−1. For picophytoplank-ton populations (Pro, Syn and P-Euks), ki was com-puted from FCM cell abundances in each dilutiontreatment. For total autotrophs (Total Phyto), dia toms,prymnesiophytes (Prym), prasinophytes (Pras), andcryptophytes (Cryp), we used changes in TChl a,Fuco, Hex-Fuco, Prasinox and Allox concentrations,respectively. The signals from minor pigments, suchas But-Fuco, DVChl a and Perid, were generally tooweak and occasional for rate calculations. The typicallinear relationship between ki and Di allowed estima-tion of m and μ daily rates from the slopes and inter-cepts of the trendlines, respectively. However, devia-tions of linearity were seen on a few occasions(mainly for the pigment data), and the μ and m ratesfor these experiments were estimated from 2 dilutiontreatments (100 and 30%) following the 2-treatmentdilution approach of Landry et al. (2008, 2009). Satu-rated grazing was also seen when a ki estimate for theundiluted treatment exceeded the estimates for the30 and 10% treatments, as described by Teixeira &Figueiras (2009). For this case, μ was calculated fromthe 10 and 30% treatments, and m was determined asthe difference between μ and the net growth rate inthe undiluted treatment (Teixeira & Figueiras 2009).Lastly, for 2 incubations, a positive relationship wasfound between Di and ki. For these, we used themeasured rates of change in the undiluted bottles,which involved no experimental mani pulation, as aminimum estimate of μ (Landry et al. 2008).

Additionally,we corrected the pigment-based growthrate estimates for photoacclimation effects during theincubations with a method adapted from Landry et al.(2003) and Gutiérrez-Rodríguez et al. (2010). Using P-Euks as a proxy for the eukaryotic phytoplanktoncommunity, we computed the ratios of P-Euks red fluorescence (F1) to forward scatter (FS) per cell from

FCM analyses (an estimate of chl a:C ratio) to reflectthe proportion of pigment change that was not due togrowth of phytoplankton carbon biomass. We usedthe change in the F1/FS ratio from initial and finalwhole seawater measurements to compute daily in-stantaneous rates of change for the 24 h (t) in -cubations as ln[(F1/FS)f /(F1/FS)i]/t. These pigment acclimation corrections were applied by subtractingthem from μ estimates based on measured TChl achanges for the total autotrophic community or from µbased on pigment-specific changes in diatoms (μFuco), prym nesiophytes (μ Hex-Fuco), prasinophytes(μ Pra si nox) and cryptophytes (μ Allox).

Primary production and grazing loss estimates

Experimental values of μ and m were combinedwith carbon biomass estimates (C0) in accordancewith Landry et al. (2000a) to compute carbon-basedestimates of production and grazing rates for the totalautotrophic community (TChl a based) and its com-ponent populations (taxon-specific cell and pigmentbased). Daily PP estimates and biomass consumptionby protistan grazers (G) expressed in carbon (µg C l−1

d−1) are computed as follows:

PP = μ (C0 [e(μ−m)t − 1]/([μ − m] × t) (1)

G = m (C0 [e(μ−m)t − 1]/([μ − m] × t) (2)

PP and G for diatoms, prymnesiophytes, prasino-phytes, cryptophytes, picoeukaryotes, Synechoccoc-cus and Prochlorococcus were estimated from theirC-based standing stocks (C0) and specific-rates(μ and m) based on Fuco, Hex-Fuco, Prasinox, Alloxpigments, as well as P-Euks, Syn and Pro cell abun-dances, respectively. Based on strong correlationbetween chlorophyll b and prasinoxanthin concen-trations (R2 = 0.90, p < 0.0001) and low ratios of luteinto chlorophyll b (Lut:chl b = 0 − 0.04; Wright & Jeffrey2006), we assumed that prasinophytes dominated thesmall green algae group. Thus, A-Flag carbon bio-mass, attributed mostly to prasinophytes, was used toestimate the dynamics of this algal taxon. Productionand grazing loss rates for other nano/ micro-sizedautotrophic eukaryotes (Other A-Euks) that couldnot be attributed to a specific phytoplankton group(mostly autotrophic dinoflagellates, A-Dino) werecomputed from rate estimates based on chl a (chl a =MVChl a + Chlide a) and total biomass for all chl a-containing autotrophs (= total biomass − Pro). Afterthe total production of the chl a-containing assem-blage was determined, the production rates for dia -toms, prymnesiophytes, prasinophytes, cryptophytes,

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P-Euks and Syn were subtracted to yield the contri-bution of Other A-Euks (Landry et al. 2011). Theratios of grazing losses to production rates (G:PP =m:μ) were estimated as measures of the daily protis-tan grazing impact on the production of the phyto-plankton community (TChl a based) and on taxon-specific phytoplankton groups (based on FCM andpigment-specific rates). For averaging, the G:PPratios for each cruise and depth level were arctan-gent transformed as in Calbet & Landry (2004).

As a basis of comparison to population productionestimates from dilution experiments, we also meas-ured primary productivity using the standard 14C-bi-carbonate-uptake technique (Steemann-Nielsen 1952).Briefly, after screening through a 150 µm net to ex-clude mesozooplankton, we inoculated the seawatersamples with ~5 µCi NaH14CO3 in 250 ml polycarbon-ate bottles. Replicated light and dark bottles wereplaced into net bags simultaneously with dilution bot-tles and incubated in situ for 24 h. Primary productionrates were calculated based on radio activity measure-ments with a Beckman LS-6500 scintillation counter,in accordance with Parsons et al. (1984).

RESULTS

Autotrophic and heterotrophic carbon biomasses

The TChl a, total autotrophic (Auto-C) and hetero-trophic (Hetero-C) carbon biomasses <200 µm aswell as relative contributions of size classes and spe-cific groups are shown in Figs. 2, 3 & 4. The totalplankton carbon biomass was highest (>150 µg C l−1)in September 2007 and April 2008 and lowest(<50 µg C l−1) during the November cruises. For allsamples, Auto-C was 58% of the total carbon bio-mass, and Hetero-C composed 42%, of which >70%was prokaryotes (Figs. 2a & 4b).

Similar trends were seen for Auto-C and TChl aduring the study period. Auto-C and TChl a valueswere highly variable in the euphotic zone, rangingfrom 4 to 192 µg C l−1 and from 0.4 to 12.5 µg chl a l−1,respectively. Extraordinary bloom levels of Auto-Cand TChl a were found in April 2008 (Fig. 2a). TheC:TChl a ratios for the phytoplankton communityaveraged (±1 SE) 24.9 ± 2.5 (Fig. 2b).

Pico- and nanoplankton were the major contributorsto the total Auto-C and Hetero-C for all cruises anddepths, except during April 2008, when total carbonwas mostly due to nano- and micro-sized cells (Fig. 3).The autotrophic picoplankton (A-Pico) biomass rangedfrom 2 to 60 µg C l−1 and was dominated by P-Euks

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Fig. 2. (a) Total autotrophic (Auto-C) and heterotrophic(Hetero-C) carbon biomass (left y-axis) and total concentra-tion of chlorophyll a (TChl a; right y-axis) and (b) Auto-C toTChl a ratios, from initial conditions of dilution experimentsconducted from September 2007 to November 2008 at

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Fig. 3. Relative contribution to total carbon biomass <200 µmof (a) autotrophic carbon (Auto-C) and (b) heterotrophic carbon (Hetero-C) by phytoplankton size class from initialconditions of dilution experiments from September 2007 toNovember 2008 at ENSENADA station. Note that hetero -trophic pico-sized cells (H-Pico) consist entirely of hetero-

trophic bacteria


and Syn (Fig. 4a). Heterotrophic pico plankton (H-Pico), composed entirely of H-Bact, ranged from 9 to70 µg C l−1 (Fig. 4b). The autotrophic nanoplankton(A-Nano) biomass, re pre sented mainly by autotrophicnano flagellates (A-Flag) and <20 µm chain-formingdiatoms, especially during September 2007 and April2008, varied from 2 to 58 µg C l−1 (Fig. 4a). The hetero-trophic nanoplankton (H-Nano) biomass, principallycomposed of heterotrophic nanoflagellates (H-Flag)and some small (<20 µm) hetero trophic dinoflagellates(H-Dino), varied from 0.4 to 7 µg C l−1 (Fig. 4b). Formicro-sized cells (autotrophic: A-Micro; heterotrophic:H-Micro), Auto-C (0.2 to 128 µg C l−1) was dominatedby dia toms and dinoflagellates (Fig. 4a), whereas Hetero-C (0.3 to 24 µg C l−1) was represented mainlyby ciliates and H-Dino (Fig. 4b).

Experimental rate estimates

Estimates of μ and m were computed for the auto -trophic community (TChl a) and for major phyto-plankton groups based on the measured net changes

in TChl a and taxon-specific pigments or FCM cellabundances, respectively (Table 1). Instantaneousgrowth and grazing rates >0 for the whole phyto-plankton community ranged from 0.24 to 1.62 d−1 andfrom 0.34 to 2.64 d−1, respectively. Mean (±1 SE) val-ues for μ and m were 0.9 ± 0.1 d−1 and 0.7 ± 0.1 d−1,respectively. Mean growth rates modestly exceededgrazing losses for most phytoplankton groups, butthe rate differences were more pronounced for largertaxa, like diatoms (Table 1).

Estimates of carbon biomass production (PP) andG based on TChl a μ and m rates for the phyto -plankton community showed marked seasonal vari-ability (Fig. 5a). Daily PP rates >0 ranged from 4 to276 µg C l−1 d−1, and G varied from 3 to 155 µg C l−1

d−1. Mean (±1 SE) values of PP and G were 55 ± 16 µgC l−1 d−1 and 36 ± 9 µg C l−1 d−1, respectively. Taxon-specific estimates of PP and G calculated for phyto-plankton groups showed variable contributions toseasonal carbon fluxes during the study period(Fig. 5b,c). Highest total production and grazinglosses were measured in the upper mixed layerduring September 2007 and April 2008 (Fig. 5a),mainly associated with larger autotrophic cells (di-atoms and Other A-Euks). P-Euks also contributednotably to production and grazing rates in September2007 (Fig. 5b,c). Low PP and G measurements weremade during cold conditions (November 2007/08 andJanuary 2008), mostly associated with smaller algalcells. Net production (= PP − G) was generally po si -tive in the ex pe rimental incubations, except for a fewcases in September 2007, January 2008 and April2008 (Fig. 5a). Additionally, production estimatesfrom the dilution experiments (TChl a based) weresimilar to net rates of 14C-uptake (Fig. 5a), whichranged from 0.24 to 217 µg C l−1 d−1, with a meanvalue of 43 ± 15 µg C l−1 d−1. Overall, the productionestimates from these 2 approaches were significantlycorrelated (R2 = 0.66, p < 0.0001).

Microzooplankton carbon consumption

Over the wide range of conditions in our study,potential grazer biomass was significantly correlatedwith autotrophic carbon biomass and with auto -trophic daily consumption rate (Fig. 6a,b). In addi-tion, estimates of autotrophic consumption (G-Auto)by protistan grazers during the study showed a tem-porally variable trophic coupling that reflectedchanges in size classes (Fig. 6c). High rates of con-sumption, mostly observed during September 2007and April 2008, were associated with higher biomass

37

Fig. 4. Relative contribution to total carbon biomass <200 µmof (a) autotrophic carbon (Auto-C) and (b) heterotrophic car-bon (Hetero-C) according to group from initial conditions ofdilution experiments from September 2007 to November 2008at ENSENADA station. A-/H-: auto/heterotrophic: Dino: dino -flagellates; Flag: flagellates; Cryp: cryptophytes; Prym: prym -nesiophytes; P-Euks: pico-eukaryotes; Syn: Synechococcus

spp.; Pro: Prochlorococcus spp.; Cil: ciliates; Bact: bacteria


of larger consumers (ciliates and flagellates >20 µm).Lower consumption rates during other periods wereassociated with assemblages of smaller grazers(Fig. 6c). Complementary shifts in protistan grazertypes that match with the dominant phytoplanktonare also suggested by significant correlations (p <0.05) between potential grazers classified by sizewith carbon biomasses and daily consumption ratesof different phytoplankton groups (Tables 2 & 3).

Grazing impact assessments on autotrophic groups

With the exception of 2 experiments in September2007 and August 2008, when growth and/or grazingrates for TChl a and consequently G:PP ratios were 0,microzooplankton grazing showed substantial protis-tan removal of autotrophic cells throughout the year(Fig. 7a). Overall, protistan consumption (based on

individual G:PP ratios) averaged 0.78 ± 0.09 for totalAuto-C (mean ± SE, n = 19). Higher predatory pres-sure (i.e. G:PP > 1) was mostly observed during earlyautumn-winter cruises (September 2007 to January2008) and in the subsurface chlorophyll maximum inApril 2008 (Fig. 7a).

For the whole study period, taxon-specific losses ofproduction to microzooplankton grazing were high-est, on average, for Pro (>100 ± 6%) followed by P-Euks (96 ± 6%), Syn (93 ± 5%), prasinophytes (87 ±10%), Other A-Euks (78 ± 13%), diatoms (45 ± 11%),prymnesiophytes (39 ± 13%) and cryptophytes (30 ±11%) (Fig. 7b,c). High predatory pressure on diatomsand Other A-Euks was mostly observed at the SCMdepth in September 2007 and April 2008. DuringJanuary 2008, both large food items and small auto -trophic prey, such as cyano bacteria, P-Euks, prym ne -sio phytes, cryptophytes and prasinophytes, werestrongly consumed at depths <20 m (Fig. 7b,c).

38

Depth Total Phyto Pro Syn P-Euks Cryp Pras Prym Diatom(m) μ m Dbl. μ m Dbl. μ m Dbl. μ m Dbl. μ m Dbl. μ m Dbl. μ m Dbl. μ m Dbl.

Sep ‘072 1.4 0.9 2.1 0.4 0.6 0.6 0.8 0.7 1.1 1.0 1.1 1.5 0.5 0.0 0.8 0.3 0.3 0.5 0.4 0.1 0.6 1.2 0.6 1.87 1.6 2.6 2.3 0.5 0.3 0.7 1.0 0.7 1.5 1.1 0.8 1.6 0.2 0.7 0.3 0.3 1.2 0.4 0.3 1.0 0.5 1.6 2.8 2.310 1.5 0.0 2.1 0.3 0.5 0.4 1.2 0.5 1.8 1.3 0.4 1.9 0.4 0.0 0.6 0.7 0.0 1.0 0.3 0.0 0.5 2.1 0.0 3.035 0.4 0.8 0.6 1.3 1.3 1.9 1.2 0.6 1.7 0.7 0.4 0.9 –0.4 0.0 na 0.0 0.0 na 0.0 0.0 na –0.2 0.0 na

Nov ‘0710 0.7 0.4 1.0 0.4 0.5 0.5 0.6 0.6 0.9 0.6 0.4 0.9 0.2 0.3 0.4 0.6 0.3 0.9 0.3 0.0 0.5 0.6 0.2 0.820 1.2 0.8 1.8 0.4 0.6 0.5 0.4 0.5 0.6 0.4 0.6 0.6 0.3 0.0 0.4 0.7 0.3 1.0 0.6 0.2 0.8 0.8 0.3 1.130 0.7 1.0 1.0 0.1 0.2 0.1 0.2 0.4 0.3 0.4 0.4 0.5 –0.1 0.0 na 0.1 0.3 0.2 0.0 0.2 0.0 0.3 0.5 0.5

Jan ‘085 0.2 0.5 0.4 0.2 0.5 0.3 0.3 0.5 0.4 0.4 0.8 0.5 –0.3 0.0 na 0.1 0.1 0.2 0.0 0.1 na 0.1 0.0 0.120 0.3 0.7 0.4 0.2 0.4 0.3 0.4 0.5 0.6 0.2 0.7 0.3 0.1 0.5 0.1 0.1 0.4 0.2 0.1 0.5 0.1 0.1 0.4 0.230 1.5 0.4 2.1 0.4 0.7 0.6 0.6 0.7 0.9 0.7 0.6 1.0 1.1 0.0 1.6 1.2 0.2 1.7 1.1 0.0 1.6 1.0 0.0 1.4

Apr ‘085 1.6 0.4 2.3 – – – 0.6 0.5 0.9 0.7 0.6 1.0 1.1 0.2 1.6 0.8 0.5 1.2 0.8 0.0 1.2 1.1 0.3 1.610 0.4 1.3 0.5 – – – 0.7 0.5 1.0 0.7 0.5 1.0 –0.1 0.7 na 0.6 0.8 0.8 –0.2 0.6 na 0.2 1.1 0.215 0.6 0.3 0.9 – – – 0.0 0.2 0.1 0.2 0.4 0.3 0.1 0.0 0.2 0.6 1.1 0.8 0.0 0.0 na 0.5 0.2 0.7

Aug ‘085 –0.1 0.0 na 0.1 0.1 0.1 0.2 0.3 0.3 0.1 0.1 0.2 –0.4 0.0 na 0.1 1.2 0.1 –0.3 0.0 na 0.1 0.0 0.110 1.2 0.7 1.8 0.9 0.5 1.3 0.7 0.5 0.9 0.4 0.3 0.6 0.5 0.0 0.7 0.9 0.5 1.3 0.7 0.2 1.0 0.8 0.3 1.120 1.0 0.7 1.5 2.1 1.5 3.0 2.1 1.4 3.0 1.5 1.2 2.2 – – – – – – – – – – – –

Nov ‘085 0.7 0.6 1.0 0.7 0.5 1.0 0.7 0.5 1.0 0.9 0.7 1.4 0.5 0.4 0.7 0.3 0.4 0.4 0.3 0.3 0.5 0.8 0.4 1.112 1.1 0.7 1.6 0.7 0.4 1.0 0.6 0.4 0.9 0.5 0.7 0.7 1.0 0.5 1.4 0.8 0.4 1.1 0.9 0.4 1.2 0.8 0.4 1.130 0.9 0.8 1.2 0.7 0.4 1.0 0.8 0.7 1.2 0.7 0.4 1.0 –0.1 0.0 na 1.6 1.2 2.3 0.2 0.1 0.3 0.1 0.0 0.1

Mean 0.9 0.7 1.4 0.6 0.6 0.8 0.7 0.6 1.0 0.7 0.6 0.9 0.3 0.2 0.7 0.5 0.5 0.8 0.3 0.2 0.7 0.7 0.4 1.0SE 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2

Table 1. Estimates of instantaneous growth (μ) rates, grazing (m) rates (d−1), doublings (Dbl.) (d−1) and averages for total phyto-plankton community (Total Phyto, based on total chl a) and for taxon-specific autotrophic groups calculated from dilutionexperiments conducted at ENSENADA station. Rates were based on cell abundances by flow cytometry analysis for Pro, Synand P-Euks. Rates were based on taxon-specific pigments for Cryp (alloxanthin-based), Pras (prasinoxanthin-based), Prym(19’-hexanoyloxyfucoxanthin-based) and diatoms (fucoxanthin-based). See Fig. 4 for abbreviations. The μ < 0, which were

approximated to 0 for average estimations, are italicized; na: not applicable


DISCUSSION

ENSENADA station is considered representativeof the dynamic physical and chemical conditions forthe northern coastal region off WBC (Linacre et al.2010b), and the 6 contrasting environmental circum-stances that we sampled during the present study area reasonable reflection of the range of variability thatcharacterizes this area. Upwelling events, which oc-cur more frequently and are stronger during springand summer in the BC region (Durazo et al. 2010), area major environmental feature of this coastal station,being associated with high nutrient delivery and en-hanced primary production. During our study, the 2

most productive periods were September 2007 andApril 2008 (Fig. 5a), when strong upwelling occurred(Linacre et al. 2010a, see their Fig. 2a). In addi -tion, most of our samplings were conducted duringLa Niña conditions from early 2007 through early summer 2008 (McClatchie et al. 2008, Durazo 2009).This cold ENSO phase brings the pycnocline and nu-tricline closer to the ocean surface, favoring entrain-ment of nutrients into the upper euphotic zone by upwelling favorable winds. During spring of 2008,this event was strongly detected at ENSENADA station, where anomalously cold (~12°C) and salty(~33.8) seawater, with low dissolved oxygen content(~150 µmol l−1) and high dissolved inorganic carbon(~2115 µmol kg−1), occurred in the upper 30 m (Li na -cre et al. 2010b). Thus, the normal seasonality of thephytoplankton community may have been especiallyintensified in April 2008 in terms of autotrophic bio-mass, community structure and production rate.

Plankton community structure

In general, the biomass of <200 µm plankton in thepresent study showed an overall close balance be-tween autotrophic and heterotrophic components(Auto-C mean ± SE = 48 ± 10 µg C l−1, Hetero-C mean± SE = 36 ± 5 µg C l−1). However, autotrophs notablydominated over heterotrophs during the in tensifiedupwelling conditions of April 2008 (Hetero-C to Auto-C ratio ~ 1:5), when carbon biomass consisted mainly(>80%) of chain-forming and large single-celled di-atoms (Figs. 2a & 4a). The up ward displacement ofupwelling water and the anomalously dense surfacewater during April 2008 could have phy si cally helpedto maintain large and heavy diatoms (some >50 µm)in the well-lit euphotic zone (Rodrí guez et al. 2001).

Similar seasonal trends have been observed inother coastal upwelling systems, where large in -creases in plankton biomass follow strong upwellingevents (Sherr et al. 2005, 2006, Vargas et al. 2007,Teixeira et al. 2011). Our Auto-C biomass values arecomparable to those in a 3 yr study conducted off ofOregon (Auto-C averaged 130 ± 170 µg C l−1 inspring-summer and 41 ± 44 µg C l−1 in winter; Sherret al. 2006), but they are lower than those found for acoastal embayment in the NW Iberian upwelling sys-tem (Total-C dominated by autotrophs ~100 µg C l−1

in winter and ~800 µg C l−1 in summer-early autumn;Teixeira et al. 2011). In this latter study, diatoms werealways present and accounted for a large fraction ofAuto-C. In contrast, diatoms played a seasonally vari-able role at our coastal site (Fig. 4a).

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Fig. 5. (a) Daily rates of primary production (PP) and grazinglosses (G ) by microzooplankton based on total concentrationof chlorophyll a (TChl a) and standard measurements of primary production from 14C uptake experiments (14C-PP).*Deviations of linearity in rate calculations. (b) Relative con-tribution to total primary production and (c) to total con-sumption rate by microzooplankton of major phytoplanktongroups based on taxon-specific cell counts or pigment con-centrations from dilution experiments conducted in the eu-photic zone from September 2007 to November 2008 at EN-SENADA station. See Fig. 4 and Table 1 for abbreviations


On average, autotrophic pico-sized cells accountedfor 57% of total phytoplankton biomass, except inApril 2008, when they comprised <10% (Fig. 3a).Similarly, H-Bact accounted for 75% of the total Hetero-C during most of our samplings (Fig. 3b).

Taylor et al. (2011) reported meaneuphotic-zone estimates of 37%(ranging from 16 to 65%) and 58% oftotal autotrophic and heterotrophiccarbon for photo trophic bacteriaand H-Bact, respectively, in easternequatorial Pacific waters. We conse-quently found biomass structure sim-ilar to the open ocean at our coastalupwelling site for most of the studyperiod. In coas tal waters off ENSE-NADA station, a large part of thevariability during summer/autumnand winter is due to the seasonalityof circulation patterns off southernCalifornia and northern BC. Amongthe patterns, surface and subsurfacewaters flow poleward in a narrowcoastal band, linked to the CaliforniaCurrent (CC) cyclonic re circulationand the permanent northern subsur-face cyclonic eddy (Li nacre et al.2010b). Thus, the meandering CCbrings waters with more oceanicinfluence (i.e. more oligotrophic con-ditions) to our coastal site by re -circulation during most of the year,carrying populations of smaller cellsbetter adapted to low-nutrient condi-tions, as suggested by a negativerelationship between log-abundanceof A-Pico cells and eupho tic zonenitrate + nitrite concentrations (Lin -acre et al. 2010a, their Fig. 10). Nutri-ent concentrations are low in the

upper 10 m during the summer/autumn and win -ter sampling periods (mostly [NO3

− + NO2−] < 2 µM

and [Si(OH)4] < 5 µM, data not shown), as expectedfor these seasons of the year in the southern CC system.

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Fig. 6. Product-moment correlation between (a) potential grazer and totalautotrophic biomasses and (b) daily rate of total autotrophic consumption andpotential grazer biomass. Dotted lines indicate 95% CI. (c) Relative initial carbonbiomass of microzooplankton grazers by size (left axis): ciliates <20 and >20 µm(Cil <20 µm and Cil >20 µm, respectively) and mixotrophic flagellates (includingdinoflagellates) <20 and >20 µm (Flag <20 µm and Flag >20 µm, respectively), inaddition to microzooplankton consumption estimates (right axis) on autotrophicorganisms (G-auto), with data estimated from dilution experiments conducted inthe euphotic zone from September 2007 to November 2008 at ENSENADA

station. Both correlations are statistically significant at p < 0.05

Total Auto-C Pro Syn P-Euks Prym Cryp A-Flag Diatom A-Dino

Total Grazers 0.84 −0.02 0.06 0.16 0.27 −0.13 0.39 0.61 0.81Nano-Flag 0.33 0.68 0.47 0.14 0.81 0.37 0.48 0.06 0.27Micro-Flag 0.83 −0.12 −0.19 −0.15 0.29 −0.18 0.21 0.66 0.98Nano-Cil −0.08 −0.02 0.33 0.31 −0.05 −0.08 0.01 −0.24 −0.14Micro-Cil 0.21 −0.09 0.23 0.49 −0.22 −0.08 0.26 0.14 −0.09

Table 2. Product-moment correlations between total and taxon-specific carbon biomasses of phytoplankton with carbon bio-masses of total potential grazers and nano/micro-flagellates (Flag <20 and >20 µm, respectively, including dinoflagellates)and nano/micro-ciliates (Cil, <20 and >20 µm, respectively), from initial samples collected during the dilution experimentsconducted from September 2007 to November 2008 at ENSENADA station. Significant correlations at p < 0.05 in bold; n = 19

(number of experiments). Abbreviations as in Fig. 4


Relatively high bacterial abundances (mean± SE = 1.29 ± 0.16 × 106 cell ml−1) and large car-bon biomasses (26.5 ± 3.9 µg C l−1) have beenfound in other coastal up welling systems. Onaverage, bacte rial abundances and biomass of1.01 ± 0.79 × 106 cells ml−1 and 30 ± 24 µg C l−1,respectively, were reported for the upper 50 min the Oregon upwelling system (Sherr et al.2006). Similarly, abundances >106 cells ml−1

and depth-integrated bacterial biomasses of1067 to 1579 mg C m−2 (our depth-integratedrange was 138 to 1123 mg C m−2) were reportedfor shallow water (<50 m depth) under gener-ally non-upwelling conditions in the HumboldtCurrent system (Cuevas et al. 2004). The largecarbon contribution of H-Bact reflects, in part,our higher cell carbon estimates (16 to 24 fg Ccell−1) compared to a more conservative esti-mate of 11 fg C cell−1 used for open-ocean stud-ies (Garrison et al. 2000, Landry & Kirchman2002, Brown et al. 2008). However, our carbonconversion factors are quite comparable toother values used in coastal ecosystems (carbonper cell from 20 to 30 fg C cell−1; e.g. Cuevas etal. 2004, Sherr et al. 2006, Teixeira et al. 2011).Thus, in comparison, the H-Bact biomass val-ues in the present study are reasonable, if notconservative.

Carbon fluxes through the microbial plankton community

Under varying physical-chemical character-istics of the water column at ENSENADA sta-tion, the community and major phytoplanktongroups displayed high variability in their pro-duction and grazing daily rates. Expressed ascarbon fluxes, these rates reflect variability in

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G-Auto G-Syn G-Pro G-P-Euks G-Prym G-Cryp G-Pras G-Diatom G-Other A-Euks (n = 19) (n = 19) (n = 16) (n = 19) (n = 18) (n = 18) (n = 18) (n = 18) (n = 16)

Total Grazers 0.50 0.04 −0.19 0.13 −0.10 −0.16 0.61 0.47 0.73Nano-Flag 0.06 0.23 0.39 0.01 0.32 0.25 0.67 −0.04 0.22Micro-Flag 0.25 −0.22 −0.15 −0.16 −0.19 −0.21 0.38 0.25 0.54Nano-Cil −0.16 0.15 −0.25 0.10 −0.06 −0.16 −0.08 −0.30 −0.33Micro-Cil 0.61 0.36 −0.32 0.51 0.03 −0.04 0.34 0.60 0.51

Table 3. Product-moment correlations between total and taxon-specific daily grazing losses of phytoplankton (G) with carbonbiomasses of total potential grazers and nano/micro-flagellates (Flag, <20 µm and >20 µm, respectively, including dinoflagel-lates) and nano/micro-ciliates (Cil, <20 µm and >20 µm, respectively) from data collected during the dilution experiments con-ducted from September 2007 to November 2008 at ENSENADA station. Significant correlations at p < 0.05 in bold; n = number

of experiments. Other A-Euks: autotrophic eukaryotes in other taxa; other abbreviations as in Fig. 4 and Table 1

Fig. 7. Arctangent ratio of daily protistan consumption (G ) to pro-duction (PP) for (a) total phytoplankton community (TChl a-based), (b) picophytoplankton groups (cell-based) and (c) majornano- and micro-sized groups (pigment-based), with data esti-mated from dilution experiments conducted from September2007 to November 2008 at ENSENADA station. Horizontaldashed lines indicate the ratio when grazing losses are equal todaily primary production (G = PP); nd: no data; see Fig. 4 and

Table 1 for other abbreviations


both the specific rates, inferred from pigment andcell-abundance measurements, and the estimatedcarbon biomasses of the phytoplankton assemblageand individual groups. Our estimates of the C:TChl aratio for the phytoplankton community were moder-ate, ranging from 5 to 41, with a mean (±1 SE) valueof 25 ± 2. This is in the range reported for othercoastal waters with similar nutrient concentrationsand light conditions (Eppley 1968, Arin et al. 2002,Gutiérrez-Rodríguez et al. 2010). Arin et al. (2002)noted that higher C:chl a ratios tend to be associatedwith larger cell size, which seems to be consistentwith our ratio estimate (~40) found in the 15 m sam-ple from April 2008 (Fig. 2a), dominated by largeautotrophic dinoflagellates (>50% of cells >20 µm;Fig. 4a). However, high C:TChla ratios were also seenduring other periods when small cells were dominant(e.g. January 2008). This seems to suggest, as inGutiérrez-Rodríguez et al. (2010), that the influenceof nutrient availability and irradiance on cellular pig-ment content prevails over cell size in our C:TChl avalues.

Comparing our dilution-based estimates of phyto-plankton production with the standard measure-ments of PP by net 14C uptake, we found a similarrange in magnitude (from 0 to 276 µg C l−1 d−1 fordilution-based PP and from 0 to 217 µg C l−1 d−1 for14C-PP), a similar seasonal variability (highest valuesin spring and lowest in winter/autumn periods) and asignificant correlation between the 2 rates (R2 = 0.66,p < 0.0001) (Fig. 5a). This close association of PP esti-mates based on different methodologies lends sup-port to our taxon-specific flux inferences, which sumto total community production.

In general, we found a close production–consump-tion coupling at our coastal site. Higher PP and G esti-mations were measured in September 2007 and April2008, associated with higher phytoplankton standingstocks and growth/grazing rates of dominant groups.Conversely, during the other periods, lower estima-tions derived from lower carbon biomasses and rateswere found in association with more oligotrophic con-ditions (Table 1, Fig. 5a). This coupled trophic patternhas also been shown for other coastal systems (Stromet al. 2001, McManus et al. 2007, Landry et al. 2009,Gutiérrez-Rodríguez et al. 2010, Teixeira et al. 2011).For such systems, it appears that adjustments in thecoupling of grazing interactions to temporal fluctua-tions of phytoplankton standing stock and productionthrough a flexible multivorous food web (herbivorousand microbial trophic modes) contribute to efficientcarbon cycling within the upper ocean (Legendre &Rassoulzadegan 1995).

Trophic coupling between carbon production andgrazing loss can be estimated among the majorautotrophic groups based on their specific contribu-tions to biogenic carbon fluxes (Fig. 5b,c). Linacre etal. (2010a) noted that auto- and heterotrophic pico -plankton generally constitute important communitycomponents at the ENSENADA station (Fig. 3).According to our estimates, A-Pico generally accountfor >50% of primary production, of which 94%, onaverage, is consumed daily by microzooplanktongrazers during most of the year (Fig. 5b,c). In spite ofseasonal variations, a close net growth/grazing bal-ance was generally found for this size category, ashas been documented for equatorial Pacific waters(Landry et al. 2011), and other coastal ecosystems(Strom et al. 2001, 2007, Worden et al. 2004). Al -though prasinophytes are an important group ofsmall green algae further to the north in the CC sys-tem according to microscopic (Thomsen & Buck1998) and molecular analyses (Worden et al. 2004,Worden 2006), they seem to contribute a minor frac-tion to carbon fluxes in the ENSENADA station.However, it is important to remark that not onlynano- but also pico-sized cells, such as Ostreococcus,belong to this group (Worden et al. 2004). Thus, someof the carbon balance attributed to P-Euks cellsmight be due to pico-prasinophytes (Fig. 5b,c).

The largest fractions of C production and C losses tograzing for both diatoms and other nano/micro-sizedautotrophic-eukaryotes (Other A-Euks, mainly dino-flagellates) occurred in April 2008 (>80% of PP and~80% of daily consumption for both large-sizedgroups), when exceptionally high values of carbonbiomass accumulation (PP > 150 µg C l−1 d−1) andlarge ciliate grazers were observed in the euphoticzone (Figs. 5a & 6c). Diatoms experienced lowerpredatory pressure by microzooplankton on averagerelative to small autotrophic cells (G:PP mean ± SE =46 ± 10%) throughout the study period, leaving abouthalf of diatom production for mesozooplankton con-sumption or export due to lateral advection or sinking.Similar microzooplankton grazing impacts on diatomswere recorded in the NW Iberian up welling system(G:PP mean ± SD = 45 ± 31%; Teixeira et al. 2011) andin the upwelling region of the Humboldt current sys-tem off Chile (25 to 45% of diatom PP during winter-autumn and 11 to 18% PP in spring-summer; Vargaset al. 2007). Total carbon and diatom losses to meso-zooplankton grazing were not estimated at our coastalstation. However, for the up welling system off south-ern California, Landry et al. (2009) found a variablecontribution of mesozooplankton relative to the micro-zooplankton grazing im pact on whole autotrophic

42


community (chl a-based) that increased in inshorewaters during springtime, suggesting strong top-down control by this size category of grazers. Thus, alarge part of diatom production that is not consumeddirectly by microherbivores at our coastal site is likelyconsumed by mesozooplankton, with more efficienttransfer to higher trophic levels (fish).

The dynamics of carbon production and grazingloss by microzooplankton in the euphotic zone of theENSENADA station indicate that biogenic carbonflows occur mainly through microbial components ofthe food web, including larger phytoplankton cellsconsumed by protists. Such multivorous food websare intermediate between the traditional herbivorousand microbial food webs. Based on Legendre & Ras-soulzadegan’s (1996) approaches that use ecologicalratios, such as small-sized (<5 µm) to large-sized(>5 µm) phytoplankton production (PPS:PPL), Mous -seau et al. (2001) evaluated planktonic food websaccording to 3 trophic pathways corresponding to theherbivorous (PPS:PPL < 1), multivorous (PPS:PPL =1.0−4.5) and microbial (PPS:PPL > 4.5, includingmicrobial loop) consumers. If we consider diatomsand Synechococcus to be representative of large andsmall autotrophs, respectively, a herbivorous path-way dominated at ENSENADA station in September2007 and April 2008 (PPS:PPL ranged from 0 to 0.9),associated with more nutrient input by the strongerand frequent upwelling events, large-sized andchain-forming diatoms and strong consumption bylarge ciliates and mixotrophic flagellates. For theother periods, trophic dominance varied from multi -vorous (mostly November cruises) to microbial path-ways, with PPS:PPL ratios oscillating between 1.3 and26.7 (with an extreme value of ~500 in August2008) in association with low nutrient concentrations,strong stratification, small primary producers andmainly nanograzer groups. These rough approxima-tions indicate that microbial components of theplanktonic food web can have significant impactson carbon fluxes during contrasting environmentalconditions in the coastal upwelling system off WBC.

Microzooplankton role in carbon fluxes

Microzooplankton play a key role as grazers of primary producers and as trophic intermediates tohigher levels of marine food webs under most envi-ronmental conditions (Calbet & Landry 2004). Assuch, the size and composition of the microzooplank-ton assemblage reflects the differing productivities ofpelagic ecosystems (Calbet 2008). Small grazers

(<20 µm), such as nanoflagellates, are the main con-sumers of phytoplankton in oligotrophic waters(Sherr & Sherr 2002, Calbet 2008), while hetero -trophic and mixotrophic dinoflagellates and ciliatesare major components in more productive waters(Neuer & Cowles 1994, Aberle et al. 2007, Sherr &Sherr 2007, Teixeira et al. 2011).

The microzooplankton consumers at our coastalsite seem to be strongly sustained by all types ofautotrophic groups (from pico to micro-sized cells), aswas evident not only from the high grazing impact(overall mean ± SE = 78 ± 9%) on the phytoplanktoncommunity and most autotrophic taxa (Fig. 7) butalso by significant correlations (p < 0.05) betweengrazer biomass, total autotrophic biomass and dailyconsumption rates (Fig. 2). Moreover, the relativeroles of micrograzers appear to follow the dominancestructure in the planktonic food web. The positiveand significant biomass correlations found betweendiatoms and A-Dino with microflagellates and alsobetween prymnesiophytes or cyanobacteria withnano flagellates (Table 2) suggest a correspondencein the size categories of autotrophic prey and theirconsumers. This is also indicated by significant corre-lations between daily consumption rates of somelarger and smaller food types with the carbon bio-mass estimates of micro- and nano-sized grazers,respectively (Table 3). Additionally, temporal vari-ability in the phytoplankton-microzooplankton linkby size category is suggested by high predatory pres-sure on pico- and nanophytoplankton (prasinophytesand prymnesiophytes) associated with the domi-nance of small ciliates and nanoflagellates in January2008, while strong grazing on diatoms was measuredin September 2007 and April 2008, when large cili-ates were major contributors to the microzooplanktonbiomass (Figs. 6c & 7b,c).

Large ciliates can be significant consumers ofdiatom chains and large single-celled diatoms incoastal waters (Aberle et al. 2007, Teixeira et al.2011). However, diatoms can also be efficiently con-sumed by heterotrophic dinoflagellates, which preyon cells as large as themselves or even larger (Sherr& Sherr 2007 and literature therein). Large pig-mented dinoflagellates, as well as other flagellates,may also be mixotrophs that contribute to grazing(Sherr & Sherr 1994, 2002). Thus, the high consump-tion of diatoms in April 2008 might not only be asso-ciated with ciliates but also with large dinoflagel-lates, such as gymnodinoids, which were particularlyevident in microscopy samples at this time. In equa-torial Pacific waters, both large dinoflagellates andciliates responded quickly to an iron-fertilized dia -

43


tom bloom (Landry et al. 2000a,b). Large dinoflagel-lates have also been reported as significant con-sumers of chain-forming diatoms in other coastal andopen-ocean systems (Neuer & Cowles 1994, Stelfox-Widdicombe et al. 2004, Teixeira et al. 2011).

Prymnesiophytes, recently found to be active mixo-trophic grazers on Prochlorococcus and Synechococ-cus in the ocean surface waters of the Pacific Ocean(Frias-Lopez et al. 2009), may also have contributedto the grazing regulation of small prey, notably inAugust 2008 and November 2008, when high con-sumption rates on Pro and high biomass and pro -duction rates for prymnesiophytes coincided (Figs. 4a& 5b,c). A significant correlation between Pro andPrym carbon biomasses (r = 0.71, p < 0.05, data notshown) was also found for our study period, suggest-ing a coupling, though not necessarily trophic, be -tween these groups. Within the trophic structure,cascade interactions among consumers could influ-ence the grazing impacts of individual size classes,e.g. by suppressing the activities of smaller grazerswhen their predators (large ciliates and dinoflagel-lates) are abundant (Calbet et al. 2008, Chen & Liu2010). In subtropical coastal waters, for example,dilution and size-fractionation experiments haverevealed trophic linkages among different micro-grazers and their small-sized prey (Chen & Liu 2010).Although a lower biomass of nanoflagellates (includ-ing small dinoflagellates) than ciliates was generallyobserved at our coastal site, we found high grazingimpacts on pico/nano-sized cells in some experi-ments in which nanoflagellate biomass was similar orever higher than that of large ciliates. Such varia -bility in composition of the grazer assemblage couldreflect temporal differences in the top-down regula-tion of large ciliates by mesozooplankton.

The present results suggest a relatively fasttrophic-coupling response in the size structure andcomposition of the microzooplankton grazer assem-blages to temporal variations of appropriately sizedprey, which would help to maintain carbon transferthrough a multivorous food web. In contrast to themore traditional herbivorous pathway that is oftenassumed for coastal upwelling systems, the multivo-rous food web promotes mineral and carbon cyclingwithin the euphotic zone. Consequently, this coastalupwelling system might be surprisingly inefficient insequestering carbon to export or in transferring pro-duction to higher trophic levels.

Acknowledgements. We gratefully acknowledge all of thestudents, technicians and scientists whose efforts facilitatedand contributed to our results, as well as the captain and

crew of RV ‘Francisco de Ulloa’ and the boat ‘GENUS’ fortheir help during the hard work at sea. A. Taylor andD. Wick contributed valuable support in training and helpwith the epifluorescence microscopy analysis and softwaredata processing. We also appreciate the valuable helpreceived from the undergraduate student P. García (FC,UABC) with ciliate counting and V. Camacho-Ibar (IIO-UABC) for the nutrient analysis. The present study was supported by FLUCAR project from CONACyT grants SEP-2004-C01-45813/A-1 and 25339, and L.L. was also sup-ported by a CONACyT fellowship (No. 201017; ReMAS2010-015). M.R.L. was supported by the California CurrentEcosystem Program (CCE-LTER; NSF OCE 04-17616 and10-22607). We are also grateful to 5 anonymous reviewersfor their valuable comments that greatly improved thiswork.

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Submitted: November 24, 2011; Accepted: April 24, 2012Proofs received from author(s): July 17, 2012

Temporal dynamics of carbon flow through the microbial plankton

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